Production of dye-decolorizing peroxidase (rDyP) from complex substrates by repeated-batch and fed-batch cultures of recombinant Aspergillus oryzae

We examined production levels of dye-decolorizing peroxidase (rDyP) by recombinant Aspergillus oryzae using wheat bran and rice bran powders in repeated-batch and fed-batch cultures. Similar average rDyP productivities were observed in repeated-batch cultures using wheat bran powder and rice bran powder. Average rDyP productivities in fed-batch cultures were slightly lower than those in repeated-batch cultures. The rDyP production was affected by the addition of K(2)HPO(4) in the repeated-batch and fed-batch cultures using wheat bran powder. All average rDyP productivities in this study were significantly higher than those for any other peroxidases previously reported.     

Effect of molasses on the production and activity of dye-decolorizing peroxidase from Geotrichum candidum Dec1

The production of dye-decolorizing peroxidase (DyP) was investigated by cultivating Geotrichum candidum Dec1 using molasses as a carbon source. Molasses at concentrations greater than 10 g.l(-1) was found to increase the decolorization activity of the culture broth toward dye, reactive blue 5 mainly because the amount of enzyme produced was enhanced. However, complete inhibition of DyP activity by molasses was observed at the concentration of 20 g.l(-1), indicating that the inhibitory effect of molasses on the culture broth activity to decolorize the dye was involved. When the culture broth was diluted 25 times, the dye-decolorizing activity was 7 times as much as that of non-diluted culture broth. The molasses fractions separated by gel chromatography (300-400 ml and 400-500 ml fractions) completely inhibited the purified DyP. On the basis of these results, we propose a scheme to control both positive and negative effects of molasses on the dye decolorization process.     

Expression of Aspergillus oryzae phytase gene in Aspergillus oryzae RIB40 niaD(-)

Aspergillus oryzae RIB40 niaD(-) was transformed using a plasmid constructed with the A. oryzae phytase gene and pNAN8142 vector. The culture broth of the transformant, which was grown in a medium containing starch as a carbon source and polyvinylpyrrolidone showed phytase activity of a maximum of 2.0 units ml(-1) at 37 degrees C, pH 5.5.     

Effect of molasses on the production and activity of dye-decolorizing peroxidase from Geotrichum candidum Dec1

The production of dye-decolorizing peroxidase (DyP) was investigated by cultivating Geotrichum candidum Dec1 using molasses as a carbon source. Molasses at concentrations greater than 10 g·l⁻¹ was found to increase the decolorization activity of the culture broth toward dye, reactive blue 5 mainly because the amount of enzyme produced was enhanced. However, complete inhibition of DyP activity by molasses was observed at the concentration of 20 g·l⁻¹, indicating that the inhibitory effect of molasses on the culture broth activity to decolorize the dye was involved. When the culture broth was diluted 25 times, the dye-decolorizing activity was 7 times as much as that of non-diluted culture broth. The molasses fractions separated by gel chromatography (300–400 ml and 400–500 ml fractions) completely inhibited the purified DyP. On the basis of these results, we propose a scheme to control both positive and negative effects of molasses on the dye decolorization process.     

Batch decolorization of molasses by suspended and immobilized fungus of Geotrichum candidum Dec 1

Geotrichum candidum Dec 1, which exhibits a broad dye-decolorizing spectrum, was used to decolorize molasses during semi-batch cultivation. An eighty % decolorization of molasses and a stable peroxidase activity were maintained for approximately four weeks, after which, both activities deteriorated significantly. Subsequently, repeated batch cultivation of fungal cells immobilized on polyurethane foam was employed to solve the aforementioned problems and stable decolorization of molasses as well as stable peroxidase activity was realized for more than eight weeks.     

Characterization of aryl alcohol oxidase produced by dye-decolorizing fungus, Geotrichum candidum Decl

Aryl alcohol oxidase (AAO) produced by Geotrichum candidum Dec1 (Dec1), a newly isolated decolorizing fungus, was purified by ultrafiltration and by using diethylaminoethyl (DEAE) Sephacel, Butyl-Toyopearl and Mono-Q columns. H2O2 produced by concomitant AAO oxidation of veratryl alcohol (VA) to veratraldehyde was consumed by a peroxidase (DyP) purified from Dec1 culture, leading to the decolorization of a dye (in vitro). In the liquid culture of Dec1, the existence of H2O2 and veratraldehyde was confirmed during cultivation, when dye-decolorization and AAO activities were maintained. This indicates that VA produced by Dec1 was oxidized by AAO to veratraldehyde, generating H2O2, which supported dye-decolorizing activity of Decl in vivo. The prevention of polymerization of DyP oxidation products of a dye in the presence of AAO was shown.     

Fed-batch coculture of Lactobacillus kefiranofaciens with Saccharomyces cerevisiae for effective production of kefiran

In a batch coculture of kefiran-producing lactic acid bacteria Lactobacillus kefiranofaciens and lactate-assimilating yeast Saccharomyces cerevisiae, lactate accumulation in the medium was observed, which inhibited kefiran production. To enhance kefiran productivity by preventing lactate accumulation, we conducted lactose-feeding batch operation with feedforward/feedback control during the coculture, so that the lactate production rate of L. kefiranofaciens was balanced with the lactate consumption rate of S. cerevisiae. The lactate concentration was maintained at less than 6 g l(-1) throughout the fed-batch coculture using a 5 l jar fermentor, although the concentration reached 33 g l(-1) in the batch coculture. Kefiran production was increased to 6.3 g in 102 h in the fed-batch coculture, whereas 4.5 g kefiran was produced in 97 h in the batch coculture. The kefiran yield on lactose basis was increased up to 0.033 g g(-1) in the fed-batch coculture, whereas that in the batch coculture was 0.027 g g(-1).     

Efficient production of a heterologous peroxidase, DyP from Geotrichum candidum Dec 1, on solid-state culture of Aspergillus oryzae RD005

The productivity of a peroxidase (DyP) originating from Geotrichum candidum Dec 1 was enhanced in the solid-state culture using Aspergillus oryzae RD005. When the humidity, water content, and temperature were adjusted to 60%, 50% and 27 degrees C, respectively, the productivity of DyP reached 5.3 g per kilogram wheat bran, which was used as the solid medium. The yield of 5.3 g per kg wheat bran corresponded to the yield of a 56 kg submerged culture. The productivity per gram carbon of the medium in the solid-state culture was 4.1-fold that in the submerged culture.     

米曲霉制备麦糟蛋白肽固态发酵培养基优化

以麦糟为原料,通过米曲霉固态发酵制备麦糟蛋白肽,研究不同培养基条件对肽转化率的影响。通过单因素实验考察碳源添加种类、碳源添加量、磷酸二氢钾添加量和料液比对肽转化率的影响,并采用响应曲面法优化培养基条件,建立肽转化率与各影响因素间的回归方程。结果表明,米曲霉发酵制备麦糟蛋白肽的最优培养基条件为:蔗糖添加量4.21%,磷酸二氢钾添加量0.51%,料液比1:3（g·mL?1）,在该条件下,发酵麦糟肽转化率可达50.73%。结果可为生物转化麦糟生产蛋白肽提供技术参考。     

玉米芯载体吸附法固定化米根霉发酵L-乳酸的工艺条件

研究玉米芯作为部分替代碳源及载体固定化米根霉R-1发酵L-乳酸的工艺条件,考察玉米芯作为替代碳源的利用率及作为载体的添加比例和最佳粉碎粒度。研究表明：米根霉R-1以玉米芯为碳源,其利用率达14.9%;玉米芯作为部分替代碳源与葡萄糖混合发酵时最佳比例为1:4,此比例下葡萄糖对L-乳酸的转化率高达82.5%,较纯葡萄糖发酵提高了14.6%;玉米芯载体的最佳粉碎粒度为40目,此条件下米根霉R-1发酵混合碳源产酸达到34.68g/L,并形成直径为0.5～1.0mm具有连续发酵能力的固定化菌丝球,连续发酵6批,葡萄糖对L-乳酸的平均转化率为83.1%。由此得出,玉米芯可以作为良好的载体及原料直接发酵产L-乳酸。     

不同高粱品种酿造酱香型白酒发酵特性的研究

通过模拟发酵实验,选用米曲霉（Aspergillus oryzae）XJDJQM03作为糖化菌株,考察菌株XJDJQM03在发酵过程对不同高粱品种淀粉的降解、利用产葡萄糖的效果,并探讨了酿酒酵母（Saccharomyces cerevisiae）YM1、YM2的发酵特性及对不同高粱品种发酵的利用。结果表明,米曲霉XJDJQM03在酿造过程对糯高粱淀粉的利用不如粳高粱,其在粳高粱样品发酵中的生物量（3.65～4.11 g/L）明显高于糯高粱样品（3.12～3.45 g/L）,贵州仁怀产红缨子糯高粱B2发酵样中的葡萄糖产量最高（168.78 mg/L）,代谢分泌水解糖化酶较多。酿酒酵母YM1对贵州仁怀产红缨子糯高粱B2发酵更具有优势,更利于酿酒酵母生长（0.80×108 CFU/mL）和代谢产乙醇（39.2 g/L）。     

烟草米根霉致病力及不同渗透压和pH环境下的代谢表型特征分析

为明确温度、叶位和创伤对烟叶霉烂病病原菌米根霉致病力的影响,及不同渗透压和pH环境下病原菌的代谢表型特征,采用穿刺法接种烟草离体叶片,测定了米根霉在不同温度及不同叶位烟叶中的致病力,并在Biolog PM 9和PM 10代谢板中96种渗透压和96种pH环境下培养米根霉,分析了其代谢表型特征.结果表明,米根霉可致病的温度...     

Growth of Aspergillus oryzae during treatment of cassava starch processing wastewater with high content of suspended solids

Aspergillus oryzae IFO 30113 was used for the treatment of the cassava starch processing (CSP) wastewater. The observations on the fungal morphology showed that, in the shake flasks containing the CSP wastewater with the high concentration of suspended solids, the formation of pellets originated from the adherence of germinated spores to solid particles in medium. The attached solid particles were also digested during the fungal fermentation and resulted in the formation of the smooth and hollow pellets. The changes of the culture conditions such as inoculum size, initial pH of wastewater, inoculum type and nutrient elements affected on the fungal morphology, biomass accumulation and treatment efficiencies of A. oryzae IFO 30113. In the typical pH range (pH 4-5) of the CSP wastewater, the formation of smooth pellets was predominant and A. oryzae IFO 30113 was satisfiable for the production of fungal biomass and treatment efficiencies. The supplementation of nitrogen sources has shown an improvement in the fungal biomass accumulation and the treatment efficiency of A. oryzae IFO 30113 growing in the CSP wastewater. Especially, high biomass yields (up to 0.8 g/g-COD) were achieved in flasks supplied with peptone. With ammonium sulfate as nitrogen source, 87% total organic carbon (TOC), 91% COD and 94% starch were removed after 96-h incubation. The possibility of the pellet formation despite the presence of the high content of suspended solids would be of great advantage to perform the treatment process and the fungal biomass production on the airlift-type bioreactors by lowering medium viscosity and better mass exchange of oxygen and nutrients.     

Abiotic and biotic factors affect efficacy of chlorfenapyr for control of stored-product insect pests

Laboratory bioassays were conducted to assess pyrole chlorfenapyr as a potential grain protectant against adults of Rhyzopertha dominica, Sitophilus oryzae, Prostephanus truncatus, Tribolium confusum, and Liposcelis bostrychophila. Factors such as dose (0.01, 0.1, 0.5, 1, 5, and 10 ppm), exposure interval (7 and 14 days), temperature (20, 25, and 30°C), relative humidity (RH; 55 and 75%), and commodity (wheat, maize, barley, and paddy rice) were evaluated. Progeny production was assessed after 74 days of exposure. For L. bostrychophila and T. confusum the increase of dose increased mortality. After 7 or 14 days of exposure, mortality was low at doses of ≤ 1 ppm and did not exceed 23 or 36%, respectively, for L. bostrychophila or 13 or 58%, respectively, for T. confusum. After 14 days of exposure, mortality of S. oryzae at 30°C and 75% RH was 82.2%. Mortality of P. truncatus was considerably higher than that of the other species. At 0.5 ppm, mortality exceeded 81% after 7 days of exposure and 91% after 14 days of exposure. Progeny production of L. bostrychophila was extremely high. Very few progeny were found for T. confusum. For S. oryzae, offspring emergence was high, except at 20°C and 55% RH. For P. truncatus, progeny production in the treated maize was not avoided, even at 10 ppm. In the case of S. oryzae, at 0.1 ppm and after 14 days of exposure, mortality in wheat was higher than in the other three commodities. For R. dominica, mortality was low at 0.1 and 1 ppm for paddy rice but reached 74.4% in barley after 14 days of exposure. For T. confusum, mortality was low at 0.1 and 1 ppm in all commodities. For progeny production counts, for S. oryzae or R. dominica, adult emergence was higher in paddy rice than in the other three commodities. Finally, overall T. confusum progeny was low. Chlorfenapyr efficacy varied remarkably among the combinations tested, and it may be a viable grain protectant in combination with other insecticides.     

Characterization of efficient aerobic denitrifiers isolated from two different sequencing batch reactors by 16S-rRNA analysis

By adopting two sequencing batch reactors (SBRs) A and B, nitrate as the substrate, and the intermittent aeration mode, activated sludge was domesticated to enrich aerobic denitrifiers. The pHs of reactor A were approximately 6.3 at DOs 2.2-6.1 mg/l for a carbon source of 720 mg/l COD; the pHs of reactor B were 6.8-7.8 at DOs 2.2-3.0 mg/l for a carbon source of 1500 mg/l COD. Both reactors maintained an influent nitrate concentration of 80 mg/l NO3- -N. When the total inorganic nitrogen (TIN) removal efficiency of both reactors reached 60%, aerobic denitrifier accumulation was regarded completed. By bromthymol blue (BTB) medium, 20 bacteria were isolated from the two SBRs and DNA samples of 8 of these 20 strains were amplified by PCR and processed for 16SrRNA sequencing. The obtained results were analysed by a Blast similarity search of the GenBank database, and constructing a phylogenetic tree for identification by comparison. The 8 bacteria were found to belong to the genera Pseudomonas, Delftia, Herbaspirillum and Comamonas. At present, no Delftia has been reported to be an aerobic denitrifier.     

复合诱变对米曲霉2336产酸量的影响

以米曲霉(Aspergillus oryzae)2336为出发菌株,经紫外线、60Co复合诱变处理,采用快速筛选法获得不产黄曲霉毒素的曲酸高产菌株UC690,以玉米淀粉为碳源,发酵7d,曲酸产量由原来的12.34g/L提高到50.47g/L。经遗传稳定性试验,这一变异菌株可应用于工业化生产。     

米曲霉液态发酵香菇残次品产蛋白酶条件优化

为了充分综合利用香菇资源，该研究以米曲霉（Aspergillus oryzae）为发酵菌种，对香菇残次品进行液态发酵，以蛋白酶活为响应值，采用单因素试验和响应面法优化米曲霉产蛋白酶发酵条件。结果表明，米曲霉产蛋白酶的最优发酵条件为发酵时间3 d、料液比1∶10（g∶mL）、初始pH值6.5、转速175 r/min，在此优化条件下，蛋白酶活力为1 182.48 U/mL。     

两株米曲霉菌种的发酵性能研究

将2株米曲霉扩大培养后用于生产试验,并将其发酵酱油的性能进行对比。结果表明,米曲霉1号酱油的氨基酸态氮、pH值、A530 nm、总氮低于米曲霉2号菌,总酸高于米曲霉2号;米曲霉2号酱油的游离氨基酸总含量为6.05 g/100 mL,是米曲霉1号（4.18 g/100 mL）的1.45倍;米曲霉1号酱油中醇类物质、酸类物质和醚类物质相对含量分别达61.53%、14.16%和1.26%,高于米曲霉2号的57.3%、13.07%和0.46%,而酯类物质（3.73%）、酮类物质（1.83%）和醛类物质（16.33%）低于米曲霉2号的3.96%、1.95%和21.31%;米曲霉1号酱油香气浓郁偏醇香味,米曲霉2号酱油香气浓郁偏酯香;米曲霉2号酱油的加热沉淀达到204 mm2,远高于米曲霉1号酱油的25 mm2。加热后过滤可去除米曲霉2号酱油中的沉淀。